The long term goal of this proposal is to define the role of protease-activated receptors (PARs) in inflammatory responses. PARs mediate cellular responses triggered by coagulation proteases and other proteases that are generated or released at sites of tissue injury. The effects of PAR activation in different cell types, largely defined in culture, suggest that PARs may help orchestrate a coordinated response to tissue injury that includes hemostasis, inflammation, and perhaps even regulation of the adaptive immune response. Local PAR activation by proteases generated at sites of local bacterial inoculation may protect against spread of bacteria by promoting both recruitment of leukocytes and microvascular thrombosis. However, more regional or systemic activation of PARs as may occur in the setting of tissue ischemia or sepsis may promote tissue damage by the same mechanisms. Toward testing these hypotheses, we have generated knockout mice for the known PARs as well as relevant double knockouts. Par4 -/- mice, for example, have no platelet responses to thrombin. Mice lacking both PAR1 and PAR4 may have lost thrombin signaling in all cell types. Using such mice and cells derived from them, we shall determine which PARs are responsible for mediating responses to coagulation proteases and other proteases in endothelial and other cell types, the extent to which different PARs in the same cell serve redundant functions, and the potential roles for endothelial PAR activation in vivo. We shall then go on to determine the effects of loss of PAR function in models of a) local bacterial infection and dissemination of same, b) systemic inflammatory response syndromes induced by endotoxin and by pancreatitis, and c) ischemia/infarction. Endpoints will include survival; markers of platelet and fibrin deposition and leukocyte accumulation in tissues; vascular permeability and edema; cytokine production; organ histology; and in a hind-limb ischemia-reperfusion model, reflow and infarct size. Lastly, because PARs sense tissue injury and hence "immunological danger," we shall ask whether PARs influence the decision to mount an adaptive immune response. These studies may point to new strategies for modulating injurious inflammatory responses in man.